Genetically Modified Cell and Process for Use of Said Cell

ABSTRACT

The present invention relates to the field of biotransformation of furanic compounds. More particular the present invention relates to novel genetically modified cells with improved characteristics for biocatalytic transformation of furanic compounds and a vector suitable for the genetic modification of a host cell. Further aspects of the invention are aimed at processes for biotransformation of 5-(hydroxymethyl)furan-2-carboxylic acid (HMF-acid) and its precursors with the use of the cell according to the invention.

FIELD OF THE INVENTION

The present invention in general relates to the field ofbiotransformation of furanic compounds. Such biotransformations findutility in the production of furan-2,5-dicarboxylic acid (FDCA) andprocessing of lignocellulose containing material e.g. for the productionof biofuels and biochemicals. More particular the present inventionrelates to novel genetically modified cells with improvedcharacteristics for biocatalytic transformation of furanic compounds. Afurther aspect of the invention relates to a vector suitable for geneticmodification of a host cell to improve its characteristics forbiotransformation of furanic compounds. Other aspects of the inventionrelate to processes for biotransformation of5-(hydroxymethyl)furan-2-carboxylic acid (HMF-acid) and its precursorswith the use of the cell according to the invention.

BACKGROUND OF THE INVENTION

Biotransformation of furanic compounds is receiving increasingattention. This is both in respect of the bioproduction offuran-2,5-dicarboxylic acid (FDCA), which is a promising value addedchemical from biomass (Werpy et al. (2004)), and in respect of theirnegative role in the fermentative production of biofuels andbiochemicals from lignocellulose containing materials (Almeida et al.(2009)).

Recently a furanic compound utilising organism, Cupriavidus basilensisHMFl4, has been isolated (Koopman et al. (2010a). This organism iscapable of metabolizing furfural and5-(hydroxymethyl)furan-2-carbaldehyde (HMF). The furfural and HMFdegradation pathway of Cupriavidus basilensis HMF14 has been disclosedby Koopman et al. (2010a) together with the genes involved.

The functional introduction of the hmfH gene from Cupriavidus basilensisHMF14 in Pseudomonas putida S12 is disclosed by Koopman et al. (2010b).The resulting strain has good FDCA production capabilities using HMF asa substrate. However, the observed accumulation of5-(hydroxymethyl)furan-2-carboxylic acid (HMF-acid) would require longprocess times or alternative measures to remove this by-product.Sufficient removal of the HMF-acid by-product is desirable for many ofthe applications for which the FDCA may be produced and sometimes evenis essential.

In search of a solution of the problem of HMF-acid accumulation, theinventors of the present invention have now surprisingly found thatexpression of certain polypeptides in the Pseudomonas putida S12 FDCAproduction system, effectively reduces HMF-acid accumulation.

SUMMARY OF THE INVENTION

The present findings of the inventors have resulted in the generalizedconcept that expression in a host of a polypeptide having an amino acidsequence of SEQ ID. NO. 1 or 2 or its analogues/homologues (such as SEQID NO: 3 or 4) together with one or more polypeptides capable ofconversion of 5-(hydroxymethyl)furan-2-carboxylic acid (HMF-acid),results in effective HMF-acid bioconversion. Improved HMF-acidbioconversion is beneficial for the elimination of HMF-acid and itsfuranic precursors from feedstocks wherein furanic compounds areconsidered to be detrimental, such as feedstocks for ethanologenicfermentations for the production of for example biofuels or forfermentations for the biological production of chemicals. In otherapplications, improved HMF-acid bioconversion will improve bioproductionof a chemical where HMF-acid is a starting material or an intermediate,such as in FDCA bioproduction.

Accordingly a first object of the invention is a genetically modifiedcell comprising a first polynucleotide sequence coding for a firstpolypeptide having an amino acid sequence of SEQ ID. NO: 1, 2, 3 or 4 orits analogues/homologues and a second polynucleotide sequence coding fora second polypeptide having HMF-acid converting activity. The HMF-acidconverting polypeptide may be the oxidoreductase encoded by theCupriavidus basilensis HMF14 hmfH gene previously described (Koopman etal. 2010a and Koopman et al. 2010b). According to certain embodiments itis preferred that the genetically modified cell comprises a thirdpolynucleotide sequence coding for a third polypeptide having an aminoacid sequence of SEQ ID. NO: 19, 20, 21, 22, 23, 24, 25 or itsanalogues/homologues. Functional expression of the third amino acidsequences results in aldehyde dehydrogenase activity capable ofconverting furanic aldehydes.

If the second polypeptide is an oxidoreductase, co-expression of thefirst polypeptide simultaneously with the oxidoreductase may alsoimprove the quality of a whole-cell biocatalyst comprising theoxidoreductase with respect to biocatalytic FDCA production.

The cell according to the invention is genetically modified byfunctional introduction of at least the first polynucleotide sequence.Preferably the cell is genetically modified by functional introductionof both the first and second polynucleotide sequence. Alternatively thecell is genetically modified by functional introduction of the first andthe third polynucleotide sequence. Functional introduction of all threeof the first, the second and the third polynucleotide is a furtheralternative. A further aspect of the invention relates to a vectorsuitable for genetic modification of a host cell. The vector comprises afirst polynucleotide sequence coding for a first polypeptide having anamino acid sequence of SEQ ID. NO. 1, 2, 3, or 4 or itsanalogues/homologues and a second polynucleotide sequence coding for asecond polypeptide having 5-(hydroxymethyl)furan-2-carboxylic acid(HMF-acid) converting activity. Optionally the vector may comprise athird polynucleotide sequence coding for a third polypeptide having anamino acid sequence of SEQ ID. NO: 19, 20, 21, 22, 23, 24, 25 or itsanalogues/homologues. Such a vector is suitable to obtain a geneticallymodified cell according to the invention.

Other aspects of the invention relate to a5-(hydroxymethyl)furan-2-carboxylic acid (HMF-acid) converting process.This process makes use of the cell according to the invention. Accordingto preferred embodiments this process is suitable for the production ofFDCA.

A further aspect of the present invention is aimed at the use of agenetically modified cell according to the invention, for thebiotransformation of furanic precursors to FDCA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 discloses a schematic representation of oxidative reactions offuranic compounds to 2.5-furan dicarboxylic acid. The following furaniccompounds are presented: 1, HMF-alcohol; 2, HMF; 3, HMF-acid; 4, FFA; 5,FDCA.

FIGS. 2a and 2b disclose FDCA production and HMF-acid accumulation inHMF-fed cultures of P. putida S12_2642 and B38 (♦, FDCA (mM); ▪,HMF-acid (mM); ◯, cell dry weight (CDW; g/l); dotted line: HMF feed rate(ml 4 M solution/h))

FIG. 3 discloses specific FDCA productivity at various time pointsduring the fed-batch processes presented in FIG. 2 (Dark: P. putidaS12_2642; Light: P. putida S12_B38).

FIGS. 4a and 4b disclose FDCA production and HMF-acid accumulation inshake-flask experiments with P. putida S12_B38 and B51 (▴, HMF (mM); ♦,FDCA (mM); ▪, HMF-acid (mM); ◯, cell dry weight (CDW; g/l));

FIG. 5 discloses FDCA production and HMF-acid accumulation in HMF-fedhigh-cell density culture of P. putida S12_B38 starting at high celldensity (□, HMF; ♦, FDCA (mM); ▪, HMF-acid (mM); ◯, cell dry weight(CDW; g/l); dotted line: HMF feed rate (ml 4 M solution/h)).

FIGS. 6a, 6b and 6c disclose the accumulation of FDCA, FFA and HMF-acidin HMF containing shake-flask cultures of P. putida S12_B38(co-expressed HmfH and HmfT1; A); S12_B97 (co-expressed HmfH, HmfT1 andaldehyde dehydrogenase; B); and S12_B101 (co-expressed HmfH and aldehydedehydrogenase; C). ▴, HMF (mM); ♦, FDCA (mM); ▪, HMF-acid (mM); •, FFA(mM); ◯, cell dry weight (CDW; g/l).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets out the amino acid sequence of HmfT1 from Cupriavidusbasilensis HMF14. The sequence has GenBank accession number ADE20411.1.

SEQ ID NO: 2 sets out the amino acid sequence of HmfT2 from Cupriavidusbasilensis HMF14. The sequence has GenBank accession number ADE20404.1.

SEQ ID NO: 3 sets out the amino acid sequence of the protein productfrom the gene with the locus tag mrad2831_4728 from Methylobacteriumradiotolerans JCM 2831 (=ATCC 27329=DSM 1819). The sequence has GenBankaccession number ACB26689.1.

SEQ ID NO: 4 sets out the amino acid sequence of the protein productfrom the saci_2058 gene from Sulfolobus acidocaldarius DSM 639. Thesequence has GenBank accession number AAY81352.1.

SEQ ID NO: 5 sets out the amino acid sequence of HmfH from Cupriavidusbasilensis HMF14. The sequence has GenBank accession number ADE20408.1.

SEQ ID NO: 6 sets out the amino acid sequence of the protein productfrom the blr0367 gene from Bradyrhizobium japonicum USDA 110. Thesequence has GenBank accession number BAC45632.1.

SEQ ID NO: 7 sets out the coding sequence of hmfT1 from Cupriavidusbasilensis HMF14.

SEQ ID NO: 8 sets out the coding sequence of hmfT2 from Cupriavidusbasilensis HMF14.

SEQ ID NO: 9 sets out the coding sequence of the gene with the locus tagmrad2831_4728 from Methylobacterium radiotolerans JCM 2831 (=ATCC27329=DSM 1819.

SEQ ID NO: 10 sets out the coding sequence of the saci_2058 gene fromSulfolobus acidocaldarius DSM 639.

SEQ ID NO: 11 sets out the coding sequence of hmfH from Cupriavidusbasilensis HMF14.

SEQ ID NO: 12 sets out the coding sequence of the blr0367 gene fromBradyrhizobium japonicum USDA 110.

SEQ ID NO: 13-18 set out the sequences of various synthetic primers.Restriction locations (underlined), start and stop (reverse complement)codons (italic) and putative ribosome binding sites (lower case) areindicated. The FN23 primer was designed just upstream of the start codonof hmfH.

SEQ ID NO: 13 sets out the nucleotide sequence of synthetic DNA primerhmfT1 (f)

5′-ACGAATTCAAaggagACAACAATGGAAG-3′

SEQ ID NO: 14 sets out the nucleotide sequence of synthetic DNA primerhmfT1 (r)

5′-AAGCTAGCTGAGCAGTCACCCTCACTC-3′

SEQ ID NO: 15 sets out the nucleotide sequence of synthetic DNA primerFN23.

5′-CGGAATTCCACATGACAagggagACCG-3′

SEQ ID NO: 16 sets out the nucleotide sequence of synthetic DNA primerFN24.

5′-CGGAATTCGCTTCGGTCTTCAACTCGGATG-3′

SEQ ID NO: 17 sets out the nucleotide sequence of synthetic DNA primermrad (f).

5′-ACGAATTCggaggAAATCTATGCAGACC-3′

SEQ ID NO: 18 sets out the nucleotide sequence of synthetic DNA primermrad (r).

5′-AAGCTAGCGCAGAACCGTATCGTCAG-3′

SEQ ID NO: 19 sets out the amino acid sequence of the aldehydedehydrogenase Adh from Cupriavidus basilensis HMF14.

SEQ ID NO: 20 sets out the amino acid sequence having Genbank accessionnumber: YP_003609156.1.

SEQ ID NO: 21 sets out the amino acid sequence having Genbank accessionnumber: ZP_02881557.1.

SEQ ID NO: 22 sets out the amino acid sequence having Genbank accessionnumber: YP_003451184.1.

SEQ ID NO: 23 sets out the amino acid sequence having Genbank accessionnumber: ACA09737.1.

SEQ ID NO: 24 sets out the amino acid sequence having Genbank accessionnumber: YP_530742.1.

SEQ ID NO: 25 sets out the amino acid sequence having Genbank accessionnumber: YP_001541929.1.

SEQ ID NO: 26 sets out the polynucleotide sequence of adh encoding thealdehyde dehydrogenase Adh from Cupriavidus basilensis HMF14.

SEQ ID NO: 27 sets out the polynucleotide sequence encoding the aminoacid sequence having Genbank accession number: YP_003609156.1.

SEQ ID NO: 28 sets out the polynucleotide sequence encoding the aminoacid sequence having Genbank accession number: ZP_02881557.1.

SEQ ID NO: 29 sets out the polynucleotide sequence encoding the aminoacid sequence having Genbank accession number: YP_003451184.1.

SEQ ID NO: 30 sets out the polynucleotide sequence encoding the aminoacid sequence having Genbank accession number: ACA09737.1.

SEQ ID NO: 31 sets out the polynucleotide sequence encoding the aminoacid sequence having Genbank accession number: YP_530742.1.

SEQ ID NO: 32 sets out the polynucleotide sequence encoding the aminoacid sequence having Genbank accession number: YP_001541929.1.

SEQ ID NO: 33 sets out the nucleotide sequence of synthetic DNA primerFN13: 5′-ATGCGGCCGCAACAaggagAAGATGGAATGAACG-3′ (underlined sequence:NotI restriction site; start codon (ATG) of aldehyde dehydrogenaseencoding gene in italics; putative ribosome binding site (RBS) in lowercase)

SEQ ID NO: 34 sets out the nucleotide sequence of synthetic DNA primerFN14: 5′-ATGCGGCCGCGCGTCGGGTCGGTGCTA-3′ (underlined sequence: NotIrestriction site, stop codon (reverse complement strand) in italics).

DETAILED DESCRIPTION OF THE INVENTION General Definitions

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle. By way of example, “an element” may mean one element or morethan one element.

The term “a number of” should be understood to have the meaning of oneor more.

Furanic compounds are herein understood to be any compound having afuran ring. Preferably the furanic compound is a compound that may beoxidized to 2,5-furandicarboxylic acid. Furanic compounds relevantwithin the context of this invention include[5-(hydroxymethyl)furan-2-yl]methanol (“HMF-alcohol”),5-(hydroxymethyl)furan-2-carbaldehyde (“HMF”),5-(hydroxymethyl)furan-2-carboxylic acid (“HMF-acid”),5-formylfuran-2-carboxylic acid (“FFA”), furan-2,5-dicarbaldehyde (DFF)and furan-2,5-dicarboxylic acid (“FDCA”). The furan ring or any of itssubstitutable side groups may be substituted, e.g., with OH, C1-C10alkyl, alkyl, allyl, aryl or RO— ether moiety, including cyclic groups,on any available position in the furan ring. The chemical structures ofa number of relevant furanic compounds are presented below.

The term “polynucleotide” includes poly deoxyribonucleic acids (DNA) andpoly ribonucleic acids (RNA) and the term may refer to either DNA orRNA. The skilled person will be aware of the differences in stability ofDNA and RNA molecules. Thus the skilled person will be able tounderstand from the context of the use of the term “polynucleotide”which of the forms of polynucleotide (DNA and/or RNA) is suitable.

The term sequence “similarity” as used herein refers to the extent towhich individual polynucleotide or protein sequences are alike. Theextent of similarity between two sequences is based on the extent ofidentity combined with the extent of conservative changes. Thepercentage of “sequence similarity” is the percentage of amino acids ornucleotides which is either identical or conservatively changed viz.“sequence similarity”=(% sequence identity)+(% conservative changes).

For the purpose of this invention “conservative changes” and “identity”are considered to be species of the broader term “similarity”. Thuswhenever, the term sequence “similarity” is used it embraces sequence“identity” and “conservative changes”.

The term “sequence identity” is known to the skilled person. In order todetermine the degree of sequence identity shared by two amino acidsequences or by two nucleic acid sequences, the sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in thesequence of a first amino acid or nucleic acid sequence for optimalalignment with a second amino or nucleic acid sequence). Such alignmentmay be carried out over the full lengths of the sequences beingcompared. Alternatively, the alignment may be carried out over a shortercomparison length, for example over about 20, about 50, about 100 ormore nucleic acids/bases or amino acids.

The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position. The degree of identityshared between sequences is typically expressed in terms of percentageidentity between the two sequences and is a function of the number ofidentical positions shared by identical residues in the sequences (i.e.,% identity=number of identical residues at corresponding positions/totalnumber of positions×100). Preferably, the two sequences being comparedare of the same or substantially the same length.

The percentage of “conservative changes” may be determined similar tothe percentage of sequence identity. However, in this case changes at aspecific location of an amino acid or nucleotide sequence that arelikely to preserve the functional properties of the original residue arescored as if no change occurred.

For amino acid sequences the relevant functional properties are thephysico-chemical properties of the amino acids. A conservativesubstitution for an amino acid in a polypeptide of the invention may beselected from other members of the class to which the amino acidbelongs. For example, it is well-known in the art of proteinbiochemistry that an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic (such as charge,hydrophobicity and hydrophilicity) can be substituted for another aminoacid without altering the activity of a protein, particularly in regionsof the protein that are not directly associated with biologicalactivity. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Conservative substitutions include, for example,Lys for Arg and vice versa to maintain a positive charge; Glu for Aspand vice versa to maintain a negative charge; Ser for Thr so that a free—OH is maintained; and Gln for Asn to maintain a free —NH₂.

For nucleotide sequences the relevant functional properties is mainlythe biological information that a certain nucleotide carries within theopen reading frame of the sequence in relation to the transcriptionand/or translation machinery. It is common knowledge that the geneticcode has degeneracy (or redundancy) and that multiple codons may carrythe same information in respect of the amino acid for which they code.For example in certain species the amino acid leucine is coded by UUA,UUG, CUU, CUC, CUA, CUG codons (or TTA, TTG, CTT, CTC, CTA, CTG forDNA), and the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU,AGC (or TCA, TCG, TCC, TCT, AGT, AGC for DNA). Nucleotide changes thatdo not alter the translated information are considered conservativechanges.

The skilled person will be aware of the fact that several differentcomputer programs, using different mathematical algorithms, areavailable to determine the identity between two sequences. For instance,use can be made of a computer program employing the Needleman and Wunschalgorithm (Needleman et al. (1970)). According to an embodiment thecomputer program is the GAP program in the Accelerys GCG softwarepackage (Accelerys Inc., San Diego U.S.A). Substitution matrices thatmay be used are for example a BLOSUM 62 matrix or a PAM250 matrix, witha gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,3, 4, 5, or 6. The skilled person will appreciate that all thesedifferent parameters will yield slightly different results but that theoverall percentage identity of two sequences is not significantlyaltered when using different algorithms.

According to an embodiment the percent identity between two nucleotidesequences is determined using the GAP program in the Accelrys GCGsoftware package (Accelerys Inc., San Diego U.S.A) A NWSgapdna CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6 is used.

In another embodiment, the percent identity of two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (Meyers et al. (1989)) which has been incorporated into theALIGN program (version 2.0) (available at the ALIGN Query using sequencedata of the Genestream server IGH Montpellier Francehttp://vega.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

For the present invention it is most preferred to use BLAST (Basic LocalAlignment Tool) to determine the percentage identity and/or similaritybetween nucleotide or amino acid sequences.

Queries using the BLASTn, BLASTp, BLASTx, tBLASTn and tBLASTx programsof Altschul et al. (1990) may be posted via the online versions of BLASTaccessible via http://www.ncbi.nlm.nih.gov/. Alternatively a standaloneversion of BLAST (e.g., version 2.2.24 (released 23 Aug. 2010))downloadable also via the NCBI internet site may be used. PreferablyBLAST queries are performed with the following parameters. To determinethe percentage identity and/or similarity between amino acid sequences:algorithm: blastp; word size: 3; scoring matrix: BLOSUM62; gap costs:Existence: 11, Extension: 1; compositional adjustments: conditionalcompositional score matrix adjustment; filter: off; mask: off. Todetermine the percentage identity and/or similarity between nucleotidesequences: algorithm: blastn; word size: 11; max matches in query range:0; match/mismatch scores: 2, −3; gap costs: Existence: 5, Extension: 2;filter: low complexity regions; mask: mask for lookup table only.

The percentage of “conservative changes” may be determined similar tothe percentage of sequence identity with the aid of the indicatedalgorithms and computer programmes. Some computer programmes, e.g.,BLASTp, present the number/percentage of positives (=similarity) and thenumber/percentage of identity. The percentage of conservative changesmay be derived therefrom by subtracting the percentage of identity fromthe percentage of positives/similarity (percentage conservativechanges=percentage similarity−percentage identity).

General molecular biological techniques such as hybridizationexperiments. PCR experiments, restriction enzyme digestions,transformation of hosts etcetera may be performed according to thestandard practice known to the skilled person as disclosed in Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols inMolecular Biology. (John Wiley & Sons, N.Y.).

First Polynucleotide and Polypeptide

The genetically modified cell according to the invention comprises afirst polynucleotide coding for a first polypeptide. The firstpolypeptide comprises an amino acid sequence having at least 45%,preferably at least 60%, such as at least 70%, more preferably at least80%, such as 90%, most preferably at least 95% sequence similarity withan amino acid sequence of SEQ ID NO: 1, 2, 3 or 4.

Alternatively the sequence similarity may be expressed as at least, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or at least 99% similarity. According to an embodiment theindicated percentages similarity may be percentages identity. In aparticular embodiment the first polypeptide may comprise the amino acidsequence as set out in any of SEQ ID NO: 1, 2, 3 or 4.

Such a polypeptide is capable of providing an improvement with respectto HMF-acid bioconversion.

Without wishing to be bound by any theory it is believed that such apolypeptide has HMF-acid transport capabilities. By transportation ofHMF-acid into the cell by the first polypeptide it becomes betteravailable for intracellular conversion. Thus HMF-acid bioconversion maybe improved.

Such an improvement on HMF-acid bioconversion has been shown in theexamples for HmfT1 (SEQ ID NO: 1) and the protein product from the genewith the locus tag mrad2831_4728 from Methylobacterium radiotolerans JCM2831 (=ATCC 27329=DSM 1819) (SEQ ID NO: 3). On the basis of the level ofsequence similarity/identity with these sequences it is justified toexpect that HmfT2 (SEQ ID NO: 2) and the protein product from theSaci_2058 gene from Sulfolobus acidocaldarius DSM 639 will have similareffects.

HmfT2 (SEQ ID NO: 2) has over 90% similarity with HmfT1 (SEQ ID NO: 1).The level of identity is 87%.

A similar functionality for the protein product of the saci_2058 genefrom Sulfolobus acidocaldarius DSM 639 (SEQ ID NO: 4) may be expectedbased on a CLUSTALW2 multiple sequence alignment with transporters fromdifferent functional families. The Sulfolobus transporter forms acluster with HmfT1 (SEQ ID NO: 1) and Mrad2831_4728 (SEQ ID NO: 3).Moreover, analysis of the S. acidocaldarius genome has shown that thetransporter gene saci_2058 is flanked by genes that encode similarfunctionalities as the hmf gene clusters, respectively, the type ofactivities that are expected in degradation of HMF(-like compounds).Examples: Saci_2057, alcohol dehydrogenase; Saci_2059/2060, aromaticring dioxygenase; Saci_2062, acyl-CoA synthetase (functionallycomparable to hmfD); Saci_2063, enoyl-CoA hydratase (functionallycomparable with hmfE); Saci_2064, aldehyde oxidase/xanthinedehydrogenase (functionally comparable to hmfABC). On the basis of thisanalysis it is justified to expect that the protein product of theSaci_2058 gene from Sulfolobus acidocaldarius DSM 639 (SEQ ID NO: 4)will have similar effects as HmfT1 (SEQ ID NO: 1) and/or the proteinproduct from the gene with the locus tag mrad2831_4728 fromMethylobacterium radiotolerans JCM 2831 (=ATCC 27329=DSM 1819) (SEQ IDNO: 3).

The first polypeptide may be encoded by a first polynucleotide sequencehaving at least 45%, preferably at least 60%, such as at least 70%, morepreferably at least 80%, such as 90%, most preferably at least 95%sequence similarity with a polynucleotide sequence set out in SEQ ID NO:7, 8, 9 or 10. Suitable alternative levels of similarity of the firstpolynucleotide with a sequence set out in SEQ ID NO: 7, 8, 9 or 10 maybe at least 66%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or at least 99% similarity. According to an embodiment theindicated percentages similarity may be percentages identity. In aparticular embodiment the first polypeptide may be coded by apolynucleotide sequence as set out in SEQ ID NO 7, 8, 9 or 10.

The first polypeptide or the polynucleotide coding for the firstpolypeptide may be isolated from an organism, preferably a microorganismthat expresses the first polypeptide under certain growth conditions.The microorganism may be capable of using HMF-acid or related furanicsubstances, such as HMF or HMF-alcohol, as carbon source, but this isnot necessary.

In a typical approach, gene libraries can be screened to isolatealternative polynucleotides which are suitable. The libraries may beconstructed from microorganisms from the superkingdom of Bacteria. Thesemicroorganisms may belong to the phylum of Proteobacteria, morespecifically to the class of Alphaproteobacteria or Betaproteobacteria.The Alphaproteobacteria may belong to the order of Rhizobiales, to thefamilies of Bradyrhizobiaceae or Methylobacteriaceae. TheBradyrhizobiaceae may belong to the genus of Bradyrhizobium, e.g.,Bradyrhizobium japonicum, or to the genus of Afipia. TheMethylobacteriaceae may belong to the genus of Methylobacterium, e.g.,Methylobacterium nodulans or Methylobacterium radiotolerans. TheBetaproteobacteria may belong to the order of Burkholderiales, morespecifically the family of Burkholderiaceae. They may belong to thegenus Cupriavidus, e.g., Cupriavidus basilensis; or to the genusRalstonia, e.g., Ralstonia eutropha; or to the genus Burkholderia, e.g.,Burkholderia phymatum, Burkholderia phytofirmans, Burkholderiaxenovorans, or Burkholderia graminis. The bacteria may also belong tothe phylum of Firmicutes, more specifically the class of Bacilli, morespecifically the order of Bacillales. The Bacillales may belong to thefamily of Bacillaceae, more specifically to the genus Geobacillus, e.g.,Geobacillus kaustophilus. Alternatively, the microorganisms may belongto the superkingdom of Archaea, more specifically the phylum ofEuryarchaeota, or the phylum of Crenarchaeota. The Euryarchaeota maybelong to an unclassified genus, e.g., Cand. Parvarchaeum acidiphilum,or to the class of Thermoplasmata, more specifically the order ofThermoplasmatales. The Thermoplasmatales may belong to the family ofThermoplasmataceae, more specifically the genus Thermoplasma, e.g.,Thermoplasma acidophilum or Thermoplasma volcanium. The Crenarchaeotamay belong to the class of Thermoprotei, more specifically the order ofSulfolobales. The Sulfolobales may belong to the family ofSulfolobaceae, more specifically the genus Sulfolobus, e.g., Sulfolobusacidocaldarius, Sulblobus islandicus, Sulfolobus solfataricus, orSulfolobus tokodaii; or to the genus of Metallosphaera, e.g.,Metallosphaera sedula. The Thermoprotei may also belong to the order ofThermoproteales, family of Thermoproteaceae. The Thermoproteaceae maybelong to the genus Vulcanisaeta, e.g., Vulcanisaeta distributa; or tothe genus Caldivirga, e.g., Caldivirga maquilingensis.

Preferably the first polypeptide and/or the first polynucleotide codingfor the first polypeptide is isolated from Cupriavidus basilensis HMF14(Wierckx et al. 2010). According to an alternative embodiment the firstpolypeptide and/or the first polynucleotide coding for the firstpolypeptide may be isolated from Methylobacterium radiotolerans.

Based on the amino acid sequences provided in SEQ. ID. NO: 1, 2, 3 or 4and/or the nucleotide sequences provided in SEQ. ID. NO: 7, 8, 9 or 10,the skilled person will be able to construct suitable probes and/orprimers to isolate a nucleotide sequence coding for the firstpolypeptide.

Alternatively, based on the amino acid sequences provided in SEQ. ID.NO: 1, 2, 3 or 4 and/or the nucleotide sequences provided in SEQ. ID.NO: 7, 8, 9 or 10, the skilled person may obtain synthesized sequencescoding for the first polypeptide from commercial sources, as genesynthesis is becoming increasingly available. Synthetic sequences may bepurchased for example from Geneart A.G. (Regensburg, Germany) or fromGenscript USA Inc. (Piscataway, N.J., USA) to name but a few.

The cell according to the invention is genetically modified byfunctional introduction of the first polynucleotide. With functionalintroduction of a polynucleotide is meant, an introduction of saidpolynucleotide in a cell, such that said cell acquires the possibilityto express a functional polypeptide product of the polynucleotide.Methods and techniques for functional introduction of polynucleotides inhost cells are within the general knowledge of the skilled person.

HMF-Acid Converting Polypeptide

The genetically modified cell according to the invention comprises asecond polynucleotide coding for a second polypeptide. The secondpolypeptide has HMF-acid converting activity and may be selected fromany polypeptide capable of converting HMF-acid to a product. Theinventors have observed that bioconversion of HMF-acid by the HMF-acidconverting second polypeptide, is effectively improved by expression ofthe first polypeptide in a cell.

The second polynucleotide coding for the second polypeptide may be anatural component of the cell according to the invention viz. the cellneed not be genetically modified in respect of the secondpolynucleotide. However, according to certain embodiments of theinvention the cell according to the invention is genetically modified inrespect of the second polynucleotide, by functional introduction of thesecond polynucleotide. The term “functional introduction” has alreadybeen explained above in connection to the first polypeptide and firstpolynucleotide.

According to a preferred embodiment of the invention, the secondpolypeptide is an HMF-acid converting oxidoreductase. This HMF-acidconverting oxidoreductase may comprise an amino acid sequence set out inSEQ ID NO: 5 or 6 or a variant polypeptide thereof having at least 45%,preferably at least 60%, such as at least 70%, more preferably at least80%, such as 90%6, most preferably at least 95% sequence similarity withthe amino acid sequence set out in SEQ ID NO 5 or 6.

Alternatively the sequence similarity may be at least, 45%, 50%6, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or at least 99% similarity. According to an embodiment the indicatedpercentages similarity may be percentages identity. In a particularembodiment the second polypeptide may comprise the amino acid sequenceas set out in any of SEQ ID NO: 5 or 6.

The HMF-acid converting oxidoreductase of SEQ ID NO: 5 was previouslydisclosed by Koopman et al. (2010a) and Koopman et al. (2010b) anddesignated HmfH. The HMF-acid converting oxidoreductase of SEQ ID NO: 6may be isolated from Bradyrhizobium japonicum USDA 110 and correspondsto the translated protein product of the blr0367 gene. B. japonicum USDA110 contains homologues for all HMF/furfural utilization genes from C.basilensis HMF14 in its genome (Koopman et al. 2010a). The translatedproduct of the blr0367 gene is the protein of B. japonicum USDA 110 thatshowed highest homology to HmfH from C. basilensis HMF14. In view of thefact that B. japonicum USDA 110 was shown to utilize HMF as the solecarbon source (Koopman et al. 2010a) it must harbour a functional HmfHhomologue. It is therefore justified to expect that HmfH similaractivity arises from blr0367.

Although the invention is exemplified with reference to a number ofHMF-acid converting oxidoreductases it should be noted that within theinvention it is expressly permitted that the second polypeptide is adifferent HMF-acid converting oxidoreductase or yet a different HMF-acidconverting polypeptide not having oxidoreductase activity.

The second polypeptide may be encoded by a second polynucleotidesequence having at least 45%, preferably at least 60%, such as at least70%, more preferably at least 80%, such as 90%, most preferably at least95% sequence similarity with a polynucleotide sequence set out in SEQ IDNO: 11 or 12. Suitable alternative levels of similarity of the firstpolynucleotide with a sequence set out in SEQ ID NO: 11 or 12 may be atleast 66%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or at least 99% similarity. In an embodiment the indicatedpercentages similarity may be percentages identity. In a particularembodiment the second polypeptide may be coded by a polynucleotidesequence as set out in SEQ ID NO 11 or 12.

Isolation of a suitable polynucleotide coding for the second polypeptidehaving oxidoreductase activity from Cupriavidus basilensis has beendisclosed in Koopman et al. (2010a). This gene is designated hmfH.

In an approach for isolation of the second polynucleotide, genelibraries can be screened to isolate polynucleotides which are suitable.The libraries may be constructed from microorganisms from thesuperkingdom of Bacteria. These microorganisms may belong to the phylumof Proteobacteria, more specifically to the class ofAlphaproteobacteria, Betaproteobacteria, or Gammaproteobacteria. TheAlphaproteobacteria may belong to the order of Rhizobiales, or the orderof Sphingomonadales. The Rhizobiales may belong to the family ofMethylobacteriaceae, e.g., an organism from the genus Methylobacteriumsuch as M. nodulans or M. radiotolerans, or an organism from the familyof Rhizobiaceae. The Rhizobiaceae may belong to theRhizobium/Agrobacterium group, more specifically to the genus Rhizobium,such as R. leguminosarum or R. leguminosarum bv. trifolii. They may alsobelong to the genus Agrobacterium, such as A. radiobacter. TheRhizobiaceae may also belong to the family of Bradyrhizobiaceae, morespecifically the genus of Bradyrhizobium, such as B. japonicum.Sphingomonedales may belong to the family of Sphingomonadaceae, morespecifically to the genus of Sphingomonas, such as S. wittichii or S.chlorophenolicum. The Betaproteobacteria may belong to the order of theMethylophilales, or the order of Burkholderiales. The Methylophilalesmay belong to the family of Methylophilaceae, e.g., an organism from thegenus Methylovorus. The Burkholderiales may belong to the family ofBurkholderiaceae, e.g., an organism from the genus Cupriavidus, such asCupriavidus basilensis. They may also belong to the genus Burkholderia,such as Burkholderia phytofirmans, B. phymatum, B. graminis, B.xenovorans, or B. cenocepacia, or to the family of Oxalobacteraceae,genus of Janthinobacterium. The Gammaproteobacteria may belong to theorder of Enterobacteriales, family of Enterobacteriaceae, genus ofYersinia such as Yersinia ruckeri. The microorganisms may furthermore bebacteria of the phylum of Actinobacteria, class of Actinobacteria,subclass of Actinobacteridae, order of Actinomycetales. TheActinomycetales may belong to the suborder of Streptomycineae,Pseudonocardineae, or Micromonosporineae. The Streptomycineae may belongto the family of Streptomycetaceae, more specifically the genusStreptomyces, such as S. violaceusniger, S. hygroscopicus, or S.clavuligerus. The Pseudonocardineae may belong to the family ofPseudonocardiaceae, more specifically the genus of Saccharopolysporasuch as S. erythraea; or to the family of Actinosynnemataceae, morespecifically to the genus of Saccharothrix such as S. mutabilis, or S.mutabilis subsp. capreolus, or to the genus Actinosynnema, such as A.mirum. The Micromonosporineae may belong to the family ofMicromonosporaceae, more specifically the genus of Micromonospora.

Based on the amino acid sequences provided in SEQ. ID. NO: 5 or 6 and/orthe nucleotide sequences provided in SEQ. ID. NO: 11 or 12, the skilledperson will be able to construct suitable probes and/or primers toisolate a nucleotide sequence coding for the second polypeptide.

Alternatively, based on the amino acid sequences provided in SEQ. ID.NO: 5 or 6 and the nucleotide sequences provided in SEQ. ID. NO: 11 or12, the skilled person may obtain synthesized sequences coding for thesecond polypeptide from commercial sources as already indicated in thesection discussing the first polypeptide.

Third Polynucleotide and Polypeptide

According to an alternative embodiment the genetically modified cellaccording to the invention comprises a third polynucleotide coding for athird polypeptide. The third polypeptide comprises an amino acidsequence having at least 45%, preferably at least 60%, such as at least70%, more preferably at least 80%, such as 90%, most preferably at least95% sequence similarity with an amino acid sequence of SEQ ID NO: 19,20, 21, 22, 23, 24 or 25.

Alternatively the sequence similarity may be expressed as at least, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or at least 99% similarity. According to an embodiment theindicated percentages similarity may be percentages identity. In aparticular embodiment the first polypeptide may comprise the amino acidsequence as set out in any of SEQ ID NO: 19, 20, 21, 22, 23, 24 or 25.The amino acid sequence of SEQ ID NO: 19 is a preferred selection of thethird polypeptide. This amino acid sequence was recently published inWO2011/026906 (SEQ ID NO: 15).

Functional expression of such a third polypeptide results in aldehydedehydrogenase activity (Adh) capable of converting furanic aldehydes andprovides a further improvement with respect to HMF-acid bioconversionand/or FDCA production.

The effects associated with the expression of the third polypeptide havebeen shown in the examples for the amino acid sequence of SEQ ID NO: 19.On the basis of the level of sequence similarity/identity it isjustified to expect that the polypeptides of SEQ ID 20-25 and theiranalogues/homologues will have similar effects.

The third polypeptide may be encoded by a third polynucleotide sequencehaving at least 45%, preferably at least 60%, such as at least 70%, morepreferably at least 80%, such as 90%, most preferably at least 95%sequence similarity with a polynucleotide sequence set out in SEQ ID NO:26, 27, 28, 29, 30, 31 or 32. Preferably the third polypeptide isencoded by a polynucleotide sequence set out in SEQ ID NO 26 or ahomologue having the indicated sequence similarity with thepolynucleotide sequence set out in SEQ ID NO: 26. The polynucleotidesequence of SEQ ID NO: 26 was recently published in WO2011/026906 (SEQID NO:16). Suitable alternative levels of similarity of the thirdpolynucleotide with a sequence set out in SEQ ID NO: 26, 27, 28, 29, 30,31 or 32 may be at least 66%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or at least 99% similarity. According to anembodiment the indicated percentages similarity may be percentagesidentity. In a particular embodiment the third polypeptide may be codedby a polynucleotide sequence as set out in SEQ ID NO 26, 27, 28, 29, 30,31 or 32.

Isolation and further manipulation of the third polypeptide and thecorresponding third polynucleotide may be performed in general as isdiscussed above and hereafter for the first polypeptide and thecorresponding first polypeptide.

Vectors

Another aspect of the invention pertains to vectors, including cloningand expression vectors, comprising the first and second polynucleotideor a functional equivalent thereof and methods of growing, transformingor transfecting such vectors in a suitable host cell, for example underconditions in which expression of a polypeptide of the invention occurs.As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked.

The first and second polynucleotide and optionally the thirdpolynucleotide, can be incorporated into a recombinant replicablevector, for example a cloning or expression vector. The vector may beused to replicate the nucleic acid in a compatible host cell. Thus in afurther embodiment, the invention provides a method of makingpolynucleotides of the invention by introducing a polynucleotide of theinvention into a replicable vector, introducing the vector into acompatible host cell, and growing the host cell under conditions whichbring about replication of the vector. The vector may be recovered fromthe host cell. Suitable host cells are described below.

The vector into which the expression cassette or polynucleotide of theinvention is inserted may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of the vectorwill often depend on the host cell into which it is to be introduced.

A vector according to the invention may be an autonomously replicatingvector, i.e. a vector which exists as an extra-chromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome (s) into which it has been integrated.

One type of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be ligated.Another type of vector is a viral vector, wherein additional DNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms “plasmid” and “vector” can be usedinterchangeably herein as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as cosmid, viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),phage vectors and transposons and plasposons, which serve equivalentfunctions.

The skilled person will be able to construct the vectors according tothe invention based on the amino acid and polynucleotide sequencesprovided, his knowledge of the art and commercially available means.

According to a preferred embodiment the first and second polynucleotidesequence are located on a single vector. This vector optionally furthercomprising the third polynucleotide sequence. As the skilled person willunderstand, the use of a vector comprising the first and secondpolynucleotide sequence (and optionally further comprising the thirdpolynucleotide sequence) greatly simplifies construction of geneticallymodified cells functionally expressing the first and second polypeptide(optionally together with the third polypeptide). However as the skilledperson will also understand, genetically modified cells functionallyexpressing the first and second polypeptide may be obtained via variousother transformation schemes involving alternative vectors. In thisrespect it should be noted that according to certain embodiments,functional introduction of the second polynucleotide sequence is not arequirement. Also according to certain further alternative embodiments,functional introduction of the third polynucleotide sequence is not arequirement.

Host Cell

The genetically modified cell according to the invention may beconstructed from any suitable host cell. The host cell may be anunmodified cell or may already be genetically modified. The cell may bea prokaryote cell, a eukaryote cell, a plant cell or an animal cell. Insuch a cell one or more genes may be deleted, knocked-out or disruptedin full or in part, wherein optionally one or more genes encode forprotease. According to an embodiment, the host cell according to theinvention is a eukaryotic host cell. Preferably, the eukaryotic cell isa mammalian, insect, plant, fungal, or algal cell. Preferred mammaliancells include, e.g., Chinese hamster ovary (CHO) cells, COS cells, 293cells, PerC6 cells, and hybridomas. Preferred insect cells include e.g.Sf9 and Sf21 cells and derivatives thereof. More preferably, theeukaryotic cell is a fungal cell, i.e., a yeast cell, such as Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia strain. More preferably, the eukaryotic cell is Kluyveromyceslactis, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowialipolytica, Pichia stipitis and Pichia pastoris, or a filamentous fungalcell. In certain embodiments, the eukaryotic cell is a filamentousfungal cell.

“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., (1995)). Thefilamentous fungi are characterized by a mycelial wall composed ofchitin, cellulose, glucan, chitosan, mannan, and other complexpolysaccharides. Vegetative growth is by hyphal elongation and carboncatabolism is obligately aerobic. Filamentous fungal strains include,but are not limited to, strains of Acremonium, Agaricus, Aspergillus,Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicilliun, Piromyces, Phanerochaete,Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia,Tolypocladium, and Trichoderma.

Preferred filamentous fungal cells belong to a species of anAspergillus, Chrysosporium, Penicillium, Talaromyces or Trichodermagenus, and most preferably a species selected from Aspergillus niger,Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae,Aspergillus fumigatus, Talaromyces emersonii, Aspergillus oryzae,Chrysosporium lucknowense, Trichoderma reesei or Penicilliumchrysogenum.

According to another embodiment, the host cell according to theinvention is a prokaryotic cell. Preferably, the prokaryotic host cellis bacterial cell. The term “bacterial cell” includes both Gram-negativeand Gram-positive microorganisms. Suitable bacteria may be selectedfrom, e.g., the genera Escherichia, Anabaena, Caulobacter,Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus,Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium),Bradyrhizobium, Flavobacterium, Klebsiella, Enterobacter, Lactobacillus,Lactococcus, Methylobacterium, Staphylococcus, Streptomyces, Zymomonas,Acetobacter, Streptococcus, Bacteroides, Selenomonas, Megasphaera,Burkholderia, Cupriavidus, Ralstonia, Methylobacterium, Methylovorus,Rhodopseudomonas, Acidiphilium, Dinoroseobacter, Agrobacterium,Sulfolobus or Sphingomonas. Preferably, the bacterial cell is selectedfrom the group consisting of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus puntis, Bacillusmegaterium, Bacillus halodurans, Bacillus pumilus, Gluconobacteroxydans, Caulobacter crescentus, Methylobacterium extorquens,Methylobacterium radiotolerans, Methylobacterium nodulans, Rhodobactersphaeroides, Pseudomonas zeaxanthinifaciens, Pseudomonas putida,Pseudomonas putida S12, Paracoccus denitrificans, Escherichia coli,Corynebacterium glutamicum, Staphylococcus carnosus, Streptomyceslividans, Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobiumradiobacter, Rhizobium leguminosarum, Rhizobium leguminosarum bv.trifolii, Agrobacterium radiobacter, Cupriavidus basilensis, Cupriavidusnecator (Ralstonia eutropha), Ralstonia picketti, Burkholderiaphytofirmans, Burkholderia phymatum, Burkholderia xenovorans,Burkholderia graminis, Rhodopseudomonas palustris, Acidiphilium cryptum,Dinoroseobacter shibae, Sulfolobus acidocaldarius, Sulfolobusislandicus, Sulfolobus solfataricus, Sulfolobus tokodaii.

A highly preferred host cell is Pseudomonas putida S12. In this strainfunctional expression of the hmfH gene from Cupriavidus basilensis HMF14has proven effective for introducing HMF oxidative capacity, resultingin FDCA production from this substrate.

For specific uses of the cell according to the invention, the selectionof the host cell may be made according to such use. Particularlypreferred are those hosts that are suitable for conversion oflignocellulosic feed stocks and those which are resistant to theconditions preferred for the production of furanic compounds, such asFDCA. The skilled person will have to his availability suitable meansand methods to functionally introduce the first and optionally thesecond poly nucleotide into any of the mentioned host cells.

HMF-acid biotransformation

The genetically engineered cell according to the invention has animproved HMF-acid biotransformation. Improved HMF-acid bioconversion isbeneficial for the elimination of HMF-acid and its furanic precursorsfrom feedstocks wherein furanic compounds are considered to bedetrimental, such as feedstocks for ethanologenic fermentations for theproduction of biofuels and biochemicals. In other applications improvedHMF-acid bioconversion will improve bioproduction of chemical whereHMF-acid is a starting material or an intermediate, such as in FDCAbioproduction.

If the HMF acid-converting enzyme is a HMF-acid convertingoxidoreductase, the cell according to the invention will be capable ofperforming a biological oxidation reaction. The oxidation reaction isherein one or more reactions of an oxidant with HMF-acid in the presenceof the oxidoreductase.

A preferred oxidation reaction is the production of FDCA, whereinHMF-acid is converted to FDCA, by reaction with an oxidant in thepresence of a HMF-acid converting oxidoreductase. Bioconversions offuranic compounds to FDCA, wherein HMF-acid is an intermediate, havebeen disclosed in the prior art, for example using HMF as a startingmaterial (see Koopman et al. 2010a and Koopman et al. 2010b). Suchbioconversions will be improved if they are performed by a cellaccording to the invention.

HMF-acid may be generated in situ from one or more furanic precursors bythe cell of the invention or any other cell present. With in situgeneration is meant that the HMF-acid is not added from outside thesystem. Instead HMF-acid is generated within the system via one or morebioconversions that convert furanic precursors to HMF-acid.

The furanic precursor of HMF-acid may be chosen from the groupconsisting of 5-(hydroxymethyl)furan-2-carbaldehyde (HMF),furan-2,5-dicarbaldehyde (DFF) and [5-(hydroxymethyl)furan-2-yl]methanol(HMF alcohol) and preferably the furanic precursor is HMF.

HMF may be obtained from one or more hexose sugars by acid-catalyzeddehydration, as is known in the art. The hexose sugars may be obtainedfrom biomass, preferably lignocellulosic biomass.

The oxidation reaction may comprise a number of consecutive oxidationreaction steps resulting in a product e.g. the oxidation of HMF-acid toFFA and further the oxidation of FFA to FDCA. Examples of oxidationreactions are given in FIG. 1.

The oxidation reactions are preferably conducted at relatively mildtemperature. i.e. 10-80° C., more preferably 20-45° C., most preferablyaround from 25-40° C. It is preferred to conduct the reaction at a pHwhere FDCA is either in a neutral form or in a fully dissociated form,such that salt formation may be controlled. In view of the presence oftwo acid moieties in FDCA there are two separate preferred pH ranges.The pH during the reaction may be from pH 1 to 6, preferably from pH 1to 4, most preferably from pH 1 to 3. Alternatively the pH during thereaction may be from pH 5 to 9, preferably from pH 5 to 8, mostpreferably from pH 5 to 7. The skilled person will understand that therequirements of the host cell will also influence the selection of asuitable pH value for the process. Selection of pH values that aresuitable for the various host cells that are suitable within the presentinvention is within the ambit of the skilled person and may be derivedfrom standard text books. For Pseudomonas putida, including Pseudomonasputida S12, the preferred pH range is from pH 5 to 7.

The reaction time may be 6-150 h, with the addition of oxygen from anoxygen source, such as molecular oxygen, or water, or a different sourceof oxygen depending on the requirements of the furanic oxidizing enzyme.Air may be used conveniently as a source of molecular oxygen.

The reactor may be any suitable (aerated) bioreactor. It may be operatedin batch, continuously or fed-batch operation.

After biotransformation, the cells may be separated from the broth byestablished methods and re-used. Oxidation products such as FDCA,HMF-acid, etc. may be recovered from the reaction mixture by (acid)precipitation and subsequent cooling crystallisation, and separation ofthe crystallized oxidation product, e.g., crystallized FDCA. However,other recovery methods are suitable, such as but not limited to acidprecipitation and solvent extraction, as known in the art.

For many applications, such as removal of HMF-acid from lignocellulosicfeedstocks, the exact way of HMF-conversion is irrelevant. What isimportant is that the HMF-acid is converted effectively in order toremove it as such, or to prevent its accumulation if it is formed fromfuranic precursors. For such applications the HMF-acid convertingpolypeptide may be any polypeptide having HMF-converting activitypresently known or yet to be discovered.

A further aspect of the present invention is aimed at the use of agenetically modified cell according to the invention, for thebiotransformation of furanic precursors to FDCA. The furanic precursorsmay in particular be selected from 5-(hydroxymethyl)furan-2-carbaldehyde(HMF), [5-(hydroxymethyl)furan-2-yl]methanol (HMF alcohol),furan-2,5-dicarbaldehyde (DFF), 5-(hydroxymethyl)furan-2-carboxylic acid(HMF-acid) or 5-formylfuran-2-carboxylic acid (FFA). Preferably HMF is aselected furanic precursor. HMF-acid may be an intermediate in thebioconversion of HMF to FDCA.

The invention will be further illustrated with reference to thefollowing examples.

EXAMPLES

General Methodology

Strains and plasmids Pseudomonas putida S12 (ATCC 700801) was used asthe host for expression of genes from Cupriavidus basilensis HMF14(Wierckx et al., 2010; Koopman et al. 2010a) and Methylobacteriumradiotolerans JCM 2831 (=ATCC 27329=DSM 1819; genome sequence availableat http://genome.jgi-psf.orgimetra/metra.home.html). Escherichia colistrains DH5α or TOP10 (Invitrogen) were used for general cloningpurposes.

For episomal expression of C. basilensis or M. radiotolerans geneseither the pUCP22-derived pJT′mcs (Koopman et al., 2010a) or pJNNmcs(t)(Wierckx et al., 2008), or the pBBR1MCS-derived pBT′mcs (Koopman et al.,2010a) was used. In pJT′mcs and pBT′mcs the expression of the targetgene is driven from the constitutive tac promoter. In pJNNmcs(t) theexpression is driven from the salicylate inducible NagR/P_(nagAa)expression cassette.

Media A Culture conditions Mineral salts medium (MM) was used as adefined medium. MM contained the following (per liter of demineralizedwater): 3.88 g of K₂HPO₄, 1.63 g of NaH₂PO₄. 2.0 g of (NH₄)₂SO₄, 0.1 gof MgCl₂.6H₂0, 10 mg of EDTA, 2 mg of ZnSO₄.7H₂0, 1 mg of CaCl₂.2H₂0, 5mg of FeSO₄.7H₂0, 0.2 mg of Na₂MoO₄.2H₂0, 0.2 mg of CuSO₄.5H₂0, 0.4 mgof CoCl₂.6H₂0, and 1 mg of MnCl₂.2H₂0, supplemented with a carbon sourceas specified. Luria broth (L-broth: 10 g/l Bacto trypton (Difco), 5 g/lyeast extract (Difco), 5 g/l NaCl) was used as a complete medium forpropagation of P. putida S12 and derivative strains, C. basilensis, M.radiotolerans and E. coli DH5α and derivatives. For plate culturing,L-broth was solidified with 1.5% (w/v) of agar (Difco). Ampicillin (Amp)was added to the media to 100 μg/ml for selection of E. colitransformants carrying pJT′mcs or pJNNmcs(t)-derived plasmids.Gentamicin (Gm) was added to 30 μg/ml in Luria broth and 10 μg/ml inmineral salts medium for selection of P. putida S12 transformantscarrying pJT′mcs or pJNNmcs(t)-derived plasmids. For selection of eitherE. coli or P. putida S12 transformants carrying pBT′mcs-derivedplasmids, 50 μg/ml of kanamycin (Km) was added to the media. Antibioticswere purchased from Sigma-Aldrich. P. putida, C. basilensis and M.radiotolerans were cultured at 30° C.; E. coli was cultured at 37° C.

Fed batch experiments with P. putida S12-derived strains were performedin 2-L vessels controlled either by a Labfors 4 Bioreactor system(Infors Benelux BV) or a BioFlo110 controller (New BrunswickScientific). Pressurized air or pure oxygen was supplied either in thehead space or sparged through the broth. The temperature was controlledat 30° C. and the pH was maintained at 7.0 by automatic addition ofeither NH₄OH, NaOH or KOH. The batch phase was performed in 2× MMmedium, supplemented with strain specific antibiotics and 40 g/lglycerol. For high-cell density cultures, the batch-phase medium wasfurthermore supplemented with 10 g/l of Yeast Extract (YE). Afterdepletion of the initial glycerol, the feed (4 or 8 M of glycerol in 100mM MgCl₂, supplemented with 1 mM of Na-salicylate when required) wasstarted and controlled to allow for growth while maintaining glycerol asthe limiting substrate in the culture. The HMF feed (4M in demineralizedwater) was fed via a separate feed pump; the feed rate was adjusteddepending on the strain employed and the condition studied. Thedissolved oxygen tension (DO) was continuously monitored and thestirring speed was adjusted to maintain sufficient aeration.

Assays & Analytical Methods Cell Dry Weight (CDW) Measurement:

CDW content of bacterial cultures was determined by measuring opticaldensity at 600 nm (OD₆₀₀) using a Biowave Cell Density Meter (WPA Ltd)or a μQuant MQX200 universal microplate spectrophotometer (Biotek),using flat-bottom 96-well microplates (Greiner). An OD₆₀₀ of 1.0corresponds to 0.56 g CDW/L (Biowave) or 1.4 g CDW/L (μQuant) for P.putida.

HPLC Analyses:

Furan compounds (FDCA, HMF, HMF-alcohol, HMF-acid and FFA) were analyzedby RP-HPLC as described by Koopman et al. (2010a). Sugars, alcohols andorganic acids were also analyzed by HPLC (Agilent 1100 system) using arefractive index (RI) detector. The column used was a Bio-Rad AminexHPX-87H (300×7.8 mm, hydrogen form, 9 μm particle size, 8% crosslinkage, pH range 1-3) with 5 mM H₂SO₄ as the eluent at a flow rate of0.6 ml/min.

Chemicals

HMF was purchased either at Sigma. Eurolabs Ltd (Poynton, UK) or YoreChemipharm Co. Ltd. (Ningbo, China). Analytical standards of FDCA and5-hydroxymethyl-furoic acid (HMF acid) were purchased from ImmunosourceB.V. (Halle-Zoersel, Belgium), respectively, Matrix Scientific (ColumbiaS.C., USA). All other chemicals were purchased from Sigma-Aldrich ChemieB.V. (Zwijndrecht, The Netherlands).

Molecular and Genetic Techniques:

Genomic DNA was isolated from C. basilensis HMF14 and M. radiotoleransJCM 2831 using the DNeasy tissue kit (QIAGEN). Plasmid DNA was isolatedwith the QIAprep spin miniprep kit (QIAGEN). Agarose-trapped DNAfragments were isolated with the QIAEXII gel extraction kit (QIAGEN).

PCR reactions were performed with Accuprime Pfx polymerase (Invitrogen)according to the manufacturer's instructions. Oligonucleotide primers(specified in the examples) were synthesized by MWG Biotech AG(Germany). Plasmid DNA was introduced into electrocompetent cells usinga Gene Pulser electroporation device (BioRad). Other standard molecularbiology techniques were performed according to Sambrook and Russell(2001).

Example I: Co-Expression of HmfH and HmfT1 Improves FDCA Production inP. putida S12

The hmfT1 gene (formerly designated mfs1 (Koopman et al., 2010a); SEQ IDNO: 7) was amplified from genomic DNA of Cupriavidus basilensis HMF14 byPCR using primers hmfT1(f) (SEQ ID NO: 13) and hmfT1(r) (SEQ ID NO: 14).The PCR product was introduced as a 1359-bp EcoRI-NheI fragment inpJNNmcs(t) yielding pJNNhmfT1 (t). The hmfH gene (SEQ ID NO: 11)including its native ribosome binding site (RBS) was amplified by PCRfrom genomic DNA of C. basilensis HMF14 using primers FN23 (SEQ ID NO15) and FN24 (SEQ ID NO: 16). The PCR product was cloned as a 1777-bpEcoRI fragment in pBT′mcs yielding plasmid pBT′hmfH. Plasmids pBT′hmfHand pJNNhmfT1(t) were successively introduced into P. putida S12,yielding P. putida S12_B38.

P. putida S12_B38 was cultured in fed-batches as described in thegeneral methodology section. In the batch phase, a cell density ofapproximately 3 g CDW/1 was achieved after which the glycerol feed andthe HMF feed were started. A control fed-batch culture was performedwith P. putida S12_2642 (similar to P. putida S12_hmfH (Koopman et al.,2010b)) which does not express HmfT1.

FIG. 2 shows the concentrations of FDCA and HMF-acid in HMF-fed culturesof P. putida strains S12_2642 and S12_B38. The extensive accumulation ofHMF-acid is evident for P. putida S12_2642. By contrast, the HMF-acidaccumulation was negligible in the P. putida S12_B38 culture. Thereduced HMF-acid accumulation furthermore allowed increased HMF feedrates, which resulted in higher FDCA titers in a considerably shorterprocess time. This was also clearly reflected in the specific FDCAproductivity for the tested strains (FIG. 3).

Example II: Co-Expression of HmfH and a Polypeptide fromMethylobacterium radiotolerans JCM 2831 Improves FDCA Production in P.putida S12

The gene with locus tag mrad2831_4728 (SEQ ID: 9) was amplified fromgenomic DNA of Methylobacterium radiotolerans JCM2831 by PCR usingprimers Mrad(f) (SEQ ID NO: 17) and Mrad(r) (SEQ ID NO: 18). The PCRproduct was introduced as a 1381-bp EcoRI-NheI fragment in pJNNmcs(t)yielding pJNN_Mrad(t). Plasmids pBT′hmfH (see Example I) andpJNN_Mrad(t) were successively introduced into P. putida S12, yieldingP. putida S12_B51.

P. putida_B51 was cultured in shake flasks on mineral salts medium (4×buffer strength) supplemented with 1 g/l yeast extract, 80 mM glycerol,2 mM glucose, 100 μM Na-salicylate, 50 μg/ml kanamycin and 10 μg/mlgentamicin. As control strain, P. putida S12_B38 was used (see ExampleI). After overnight culturing, the cultures were supplemented with 80 mMglycerol and 100 μM Na-salicylate. Subsequently, approximately 10 mM HMFwas added and FDCA production was assessed.

FIG. 4 shows that the HMF-acid accumulation during FDCA production wasnegligible for both strains, confirming that HmfT1 and the Mrad2831_4728polypeptide exhibited a similar functionality. For P. putida S12_B38,the FDCA production showed a longer lag phase and a somewhat slowerrate, which could be attributed to the lower initial biomass density(FIG. 4A). The specific maximum FDCA productivity, however, wasidentical for both strains, i.e., 2.36 mmol FDCA/(g CDW, h), indicatingthat the Mrad2831_4728 and HmfT1 polypeptides were equally effective inminimizing HMF-acid accumulation and maximizing FDCA production.

Example III: High-Level HMF-Acid Conversion Capacity by P. putida S12Co-Expressing HmfH and HmfT1

As demonstrated in Example I, co-expression of HmfH and HmfT1 in P.putida S12 considerably improved the specific capacity to oxidize HMF toFDCA. To make optimal use of this improved capacity, a fed-batchexperiment was performed with P. putida S12_B38 starting at a highbiomass density.

The HMF feed was started at a high rate (20 ml/h; 4 M HMF feed solution)in order to saturate the oxidation capacity of P. putida S12_B38 andprovoke the accumulation of HMF and HMF-acid (FIG. 5). When HMF-acid hadaccumulated to approximately 20 mM, the HMF feed rate was lowered to 5ml/h and the furanics concentrations were monitored. Initially, theHMF-acid concentration increased to approximately 37 mM due to oxidationof residual accumulated HMF, after which it dropped to less than 2 mMwithin 5 h, at an HMF feed rate of 0.72 mmol/(g CDW·h).

These results clearly demonstrate that the HMF-acid oxidation capacitywas improved by co-expression of HmfH and HmfT1. The results by Koopmanet al. (2010b), showed that P. putida S12_hmfH (which lacks HmfT1)required over 50 h to reduce the HMF-acid concentration fromapproximately 50 mM to less than 5 mM, at a much lower HMF feed rate(0.09 mmol/(g CDW·h)). The improved HMF-acid oxidation capacity resultedin a much higher final FDCA titer (152 g/l vs 30.1 g/l by Koopman et al.(2010b)) that was furthermore achieved in a shorter process time (94 hvs 115 h by Koopman et al. (2010b)).

Example IV: Co-Expression of HmfH, HmfT1 and an Aldehyde Dehydrogenasefrom C. basilensis HMF 14 Improves FDCA Production in P. putida S12

The gene encoding an aldehyde dehydrogenase (SEQ ID 26; translated aminoacid sequence: SEQ ID 19) associated with the HMF-degradation operon inCupriavidus basilensis HMF14 (Wierckx et al., 2011) was amplified by PCRusing primers FN13 (SEQ ID 33) and FN14 (SEQ ID 34). The PCR product wasintroduced as a 1543-bp NotI fragment in Bsp120I-digested (compatible toNotI) pBT′hmfH (see example I) yielding pBT′hmfH-adh. The plasmidvariant in which the aldehyde dehydrogenase encoding gene was present inthe forward (f) orientation (pBT′hmfH-adh(f)) and pJNNhmfT1(t) (seeexample I) were successively introduced into P. putida S12. Theresulting strain, P. putida S12_B97, co-expressed HmfH, HmfT1, and thealdehyde dehydrogenase. As a control strain for co-expression of theHmfH oxidoreductase and the aldehyde dehydrogenase (i.e., without theHMF-acid transporter HmfT1), P. putida S12_B101 was constructed whichonly contained pBT′hmfH-adh(f). P. putida S12_B38 (see example I) wasused as control strain for co-expression of HmfT1 and HmfH without thealdehyde dehydrogenase.

P. putida strains S12_B38, S12_B97 and S12_B101 were cultured in shakeflasks on mineral salts medium (4× buffer strength) supplemented with 1g/l yeast extract, 80 mM glycerol, 2 mM glucose, 50 g/ml kanamycin and10 μg/ml gentamicin (note: for strain B101 only kanamycin was added).Na-salicylate (1 μM) was added for induction of hmfT1 in the preculturesonly. After addition of approximately 10 mM HMF, the accumulation ofFDCA and HMF-acid was assessed.

In the strain that co-expressed HmfH (oxidoreductase) and HmfT1(HMF-acid transporter) (strain S12_B38; FIG. 6A), FDCA production onlycommenced after FFA had accumulated to a substantial level. HMF acidaccumulated transiently to low amounts, as observed previously (seeexample II). When the aldehyde dehydrogenase was co-expressed with HmfHand HmfT1 (strain S12_B97; FIG. 6B), FDCA formation commenced withoutdelay, and both FFA and HMF-acid were observed only in trace amounts.Co-expression of the aldehyde dehydrogenase and HmfH without HmfT1(strain S12_B101; FIG. 6C), resulted in extensive accumulation ofHMF-acid whereas only small amounts of FFA and FDCA were produced.

The results demonstrated that the oxidation of HMF to HMF-acid issignificantly enhanced by expressing the aldehyde dehydrogenase. HmfT1must be co-expressed, however, to enable efficient biotransformation ofthe HMF-acid produced. The aldehyde dehydrogenase furthermore improvedthe oxidation of the intermediate product FFA to FDCA. Thus,simultaneous expression of the aldehyde dehydrogenase and HmfT1considerably improves the overall potential for, and rate of, HMFoxidation via HMF-acid to the final product.

REFERENCES

-   Almeida et al. (2009) Metabolic effects of furaldehydes and impacts    on biotechnological processes. Applied Microbiology and    Biotechnology, 82 (4); 625-638.-   Altschul et al., (1997) Gapped BLAST and PSI-BLAST: a new generation    of protein database search programs. Nucleic Acids Res, 25 (17):    3389-3402.-   Altschul, et al., (1990) Basic local alignment search tool. J. Mol.    Biol., 215: 403-10.-   Ausubel et al. (eds.) (1995), Current Protocols in Molecular    Biology, (John Wiley & Sons, N.Y.-   Hawksworth et al., (1995) In: Ainsworth and Bisby's Dictionary of    The Fungi, 8th edition, CAB International, University Press,    Cambridge, UK.-   Koopman et al., (2010a) Identification and characterization of the    furfural and 5-(hydroxymethyl)furfural degradation pathways of    Cupriavidus basilensis HMF14. PNAS (2010a), Vol. 107, p. 4919-4924.-   Koopman et al., (2010b) Efficient whole-cell biotransformation of    5-(hydroxymethyl)furfural into FDCA, 2,5-furandicarboxylic acid.    Bioresour. Technol. (2010b), Vol. 101, p. 6291-6296.-   Meyers et al., (1988) Optimal alignments in linear space CABIOS,    4:11-17.-   Needleman et al., (1970) A general method applicable to the search    for similarities in the amino acid sequences of two proteins. J.    Mol. Biol. (48): 444-453.-   Nichols N N, Mertens J A. Identification and transcriptional    profiling of Pseudomonas putida genes involved in furoic acid    metabolism (2008) FEMS Microbiol Lett 284: 52-57-   Sambrook et al., (1989), Molecular Cloning, A Laboratory Manual,    Cold Spring Harbor Press, N.Y.-   Sambrook et al., (2001) Molecular cloning—a laboratory manual. Third    edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,    N.Y.-   Werpy et al., (2004) Top Value-Added Chemicals from Biomass, Volume    I—Results of screening for potential Candidates from Sugars and    Synthesis gas.-   Wierckx et al., (2008) Transcriptome analysis of a phenol producing    Pseudomonas putida S12 construct: genetic and physiological basis    for improved production. J Bacteriol 190: 2822-2830.-   Wierckx et al. (2010) Isolation and characterization of Cupriavidus    basilensis HMF14 for biological removal of inhibitors from    lignocellulosic hydrolysate. Microb Biotechnol 3: 336-343.-   Wierckx et al., (2011) Microbial degradation of furanic compounds:    biochemistry, genetics, and impact. Appl Microbiol Biotechnol    92:1095-1105

1-24. (canceled)
 25. A process for producing 2,5-furandicarboxylic acid(FDCA), the process comprising the step of incubating a cell in thepresence of one or more furanic precursors of HMF-acid under conditionssuitable for the oxidation by said cell of the one or more furanicprecursors to FDCA, wherein said conditions include that the cell isincubated at a pH from pH 1 to 6, and wherein the cell is geneticallymodified to express a polynucleotide sequence coding for a polypeptidehaving HMF-acid oxidoreductase activity.
 26. The process of claim 25,wherein the cell is incubated at a pH from pH 1 to
 4. 27. The process ofclaim 26, wherein the cell is incubated at a pH from pH 1 to
 3. 28. Theprocess of claim 25, wherein the cell is a fungal cell.
 29. The processof claim 28, wherein the cell belongs to a fungal genus selected fromthe group consisting of the genera Candida, Hansenula, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia, Acremonium,Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus,Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Piromyces, Phanerochaete, Pleurotus, Schizophyllum, Talaromyces,Thermoascus, Thielavia, Tolypocladium and Trichoderma.
 30. The processof claim 29, wherein the cell is of a fungal species selected from thegroup consisting of Kluyveromyces lactis, Saccharomyces cerevisiae,Hansenula polymorpha, Yarrowia lipolytica, Pichia stipitis, Pichiapastoris, Aspergillus niger, Aspergillus awamori, Aspergillus foetidus,Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii,Aspergillus oryzae, Chrysosporium lucknowense, Trichoderma reesei andPenicillium chrysogenum.
 31. The process of claim 25, wherein thepolypeptide having HMF-acid oxidoreductase activity comprises an aminoacid sequence having at least 70% sequence identity with at least one ofSEQ ID NO: 5 and
 6. 32. The process of claim 25, wherein the cell isfurther genetically modified cell to express a polynucleotide sequencecoding for a polypeptide having furanic aldehyde dehydrogenase activity.33. The process of claim 32, wherein the polypeptide having furanicaldehyde dehydrogenase activity comprises an amino acid sequence havingat least 45% sequence identity with at least one of SEQ ID NO: 19, 20,21, 22, 23, 24 and
 25. 34. The process of claim 25, wherein the at leastone furanic precursor is selected from the group consisting of5-(hydroxymethyl)furan-2-carbaldehyde (HMF), furan-2,5-dicarbaldehyde(DFF), [5 (hydroxymethyl)furan-2-yl]methanol (HMF alcohol).
 35. Theprocess of claim 34, wherein the furanic precursor is obtained from oneor more hexose sugars.
 36. The process of claim 35, wherein the furanicprecursor is obtained from one or more hexose sugars by acid-catalyzeddehydration.
 37. The process of claim 25, wherein FDCA is recovered by aprocess comprising acid precipitation.
 38. The process of claim 37,wherein the acid precipitation is followed by at least one of coolingcrystallization and solvent extraction.